Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin.

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 µg ml-1 while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.

Piper regnellii extract biopolymer-based microparticles: production, characterization and antifungal activity.

AIMS: This study aims to improve characteristics of Piper regnellii extract to make it applicable in formulations to treat dermatophytosis, also known as ringworm. METHODS AND RESULTS: Microparticles (MPs) were produced by spray drying with gelatin, alginate and chitosan as encapsulating agents; characterized by scanning electron microscopy, encapsulation efficiency, thermal analyses and X-ray diffraction; and tested against Trichophyton rubrum by broth microdilution. Produced MPs had a mean diameter less than 2 µm, an increase in stability and release of the extract and good results for encapsulation efficiency, being 85·6% to gelatin MP, 71·3% to chitosan MP and 60·6% to alginate. MPs preserved the antifungal activity of P. regnellii extract T. rubrum. CONCLUSION: Microencapsulation provided a significant improvement in the stability of the P. regnellii extract and better solubilization of chemical compounds, maintaining the antifungal effect against T. rubrum. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are useful for developing a formulation to treat fungal infections caused by dermatophyte species.

Beta-carboline-3-carboxamide derivatives as promising antileishmanial agents.

Leishmaniasis has an overwhelming impact on global public health especially in tropical and subtropical countries and the currently available antileishmanial drugs have serious side effects and low efficacy. Natural and synthetic compounds have been tested in the past few years against Leishmania and the beta-carboline class of compounds have shown great results in antiparasitic chemotherapy. In the present study, three 1-substituted beta-carboline-3-carboxamides (3-5) and 1-substituted beta-carboline-3-carboxylic acid (2) were synthesized and screened for in vitro activity against L. amazonensis. Compound 5 (N-benzyl 1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide) had the best activity against promastigote and axenic amastigote forms with IC(50) of 2·6 and 1·0 µM, respectively. Its CC(50) on macrophages cell line was higher than 2457·0 µM with an SI ratio of 930·2. Against intracellular amastigote forms, it had a dose-dependent relationship with a 50% growth inhibitory concentration of 1·0 µM. Through morphological and ultrastructure analysis of promastigote forms treated with compound 5, alterations on cell shape and number of flagella and nuclear membrane damage were observed. For this, compound 5 supports the idea for more in vitro and in vivo studies.

In vitro anti-trypanosomal activity of elatol isolated from red seaweed Laurencia dendroidea.

SUMMARY: Chagas' disease is a debilitating but comparatively neglected illness that affects about 15 million people. There is an urgent need to develop new, more effective, and less-toxic compounds. In this study, we assessed the in vitro anti-trypanosomal activity of the sesquiterpene elatol from the Brazilian red seaweed Laurencia dendroidea. We used electron microscopy to evaluate the effect of elatol on the morphology and ultrastructure of the parasite. Elatol showed a dose-dependent effect against the epimastigote, trypomastigote, and amastigote forms, with IC50 values of 45.4, 1.38, and 1.01 microm, respectively. Observation of treated intracellular amastigotes by light microscopy demonstrated a total elimination of the infection at a dose of 3.0 microm. In addition, the compound did not affect the red blood cells, and the CC50 value for LLCMK2 cells was 27.0 microm. Transmission and scanning electron micrographs showed aberrant-shaped cells and breaks in the plasma membrane, prominent swollen mitochondria, and extensive formation of cytoplasmic vacuoles in all the forms. This is the first report of the anti-trypanosomal effect of the sesquiterpene elatol.

Antileishmanial activity of an essential oil from the leaves and flowers of Achillea millefolium.

An essential oil was recently extracted from the leaves and flowers of yarrow (Achillea millefolium) and tested for in-vitro activity against Leishmania amazonensis and murine macrophages (i.e. the J774G8 cell line). The median inhibitory concentration (IC(50)) against L. amazonensis promastigotes was 7.8 µg/ml whereas the survival of amastigotes of this pathogen, within peritoneal murine macrophages, was halved by treatment with the oil at 6.5 µg/ml. The mean value for the median cytotoxic concentration of the oil, measured against adherent (uninfected) J774G8 macrophages, was 72.0 µg/ml (i.e. 9.2 and 11.0 times higher, respectively, than the IC(50) against the promastigotes and intracellular amastigotes). Scanning electron microscopy revealed that the oil caused morphological changes in the treated parasites, including alterations in their shape and size. In transmission electron microscopy, promastigotes treated with the oil (at the IC(50) of 7.8 µg/ml) showed various ultrastructural alterations, including changes in the flagellar membrane, abnormal membrane structures, rupture of the plasma membrane, atypical vacuoles, myelin-like figures, and vesicles that resembled autophagic vacuoles.

Ketoconazole-loaded poly-(lactic acid) nanoparticles: Characterization and improvement of antifungal efficacy in vitro against Candida and dermatophytes.

OBJECTIVE: In order to improve the effect of ketoconazole, poly-lactic acid (PLA) nanoparticles containing ketoconazole were prepared, characterized and tested against dermatophytes and Candida spp planktonic and biofilm cells. METHODS: The ketoconazole-PLA nanoparticles obtained by nanoprecipitation were characterized using dynamic light scattering, scanning electron microscopy, transmission electron microscopy, and differential scanning calorimetry. In addition, quantification of encapsulated ketoconazole and the in vitro release profile were determined. Antifungal susceptibility tests against dermatophytes Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum and yeasts Candida albicans, C. dubliniensis, C. krusei, C. parapsilosis, and C. tropicalis were performed. RESULTS: Spherical nanoparticles, with a mean diameter of 188.5nm and an encapsulation efficiency of 45% ketoconazole, were obtained. The nanoparticles containing ketoconazole had superior antifungal activity against all tested fungi strains than free ketoconazole. Inhibition of yeast biofilm formation was also achieved. CONCLUSION: Ketoconazole-PLA nanoparticles resulted in better antifungal activity of ketoconazole nanoparticles than free drug against dermatophytes and Candida species, indicating a promising tool for the development of therapeutic strategies.

Preparation, characterization and antidermatophytic activity of free- and microencapsulated cinnamon essential oil.

Essential oils (EO) are effective natural antimicrobials but are susceptible to oxidation. Microencapsulation improves EO stability, reduces toxicity, and controls release. The aim of this study was preparation, characterization and antidermatophytic activity of free and microencapsulated cinnamon essential oil (MP). MP were prepared by the spray drying method and the success of MP encapsulation was confirmed by UV-vis spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. The antifungal effect of EO and MP was evaluated by the broth microdilution method against Microsporum gypseum and Trichophyton mentagrophytes. The checkerboard method was used to assess synergistic interactions. Fluorescence microscopy and scanning electron microscopy were used to investigate the inhibition of hyphal growth by EO and MP. A cytotoxic assay was performed using the VERO cell line. Microencapsulated cinnamon essential oil was found to be micrometric, with a round, regular structure. The minimum inhibitory concentration of EO was found to be between 125-250µg/mL, while that of MP was 220.5-440.5µg/mL. EO was synergistic with fluconazole while microencapsulated oil was less cytotoxic than EO.

Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum.

We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10(7) CFU/mL). Microtiter plates were incubated at 37 masculineC and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 microg/mL) and S. aureus (MIC = 500 microg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 microg/mL). Fractions I and II were inactive against standard strains at concentrations of < or =1000 microg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 microg/mL) and S. aureus (MIC = 250 microg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 microg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 microg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 microg/mL) and S. aureus (MIC = 31.3 microg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 microg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 microg/mL) and E. faecalis (MIC = 50 microg/mL), with EC50 of 8 and 2.5 microg/mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

Antimicrobial effects of Piper hispidum extract, fractions and chalcones against Candida albicans and Staphylococcus aureus.

Three chalcones, 2'-hydroxy-4,4',6'-trimethoxychalcone, 2'-hydroxy-4,4',6'-tetramethoxychalcone, and 3,2'-dihydroxy-4,4',6'-trimethoxychalcone, were isolated from the leaves of Piper hispidum in a bioguided fractionation of crude extract. The antimicrobial activity of crude extract of P. hispidum leaves was determined against bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and yeasts Candida albicans, C. parapsilosis and C. tropicalis. Fractions and chalcones were tested against C. albicans and S. aureus. The checkerboard assay was performed to assess synergic interactions between extract and antifungal drugs, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was used to evaluate anti-biofilm effects of extract. The extract was active against yeasts, S. aureus and B. subtilis with MIC values between 15.6 and 62.5µg/mL. Synergistic effects of extract associated with fluconazole and nystatin were observed against C. albicans, with fractional inhibitory concentration indices of 0.37 and 0.24, respectively. The extract was also effective against C. albicans and S. aureus biofilm cells at concentrations of 62.5 and 200µg/mL, respectively. Thus, P. hispidum may be a possible source of bioactive substances with antimicrobial properties.

Antidermatophytic activity of hydroalcoholic extracts from Rosmarinus officinalis and Tetradenia riparia.

Rosmarinus officinalis and Tetradenia riparia are used in folk medicine for the treatment of disease, including infectious diseases and skin disorders. The purpose of this study was to investigate the antifungal activity of hydroalcoholic extracts from R. officinalis and T. riparia against strains of Trichophyton rubrum, T. mentagrophytes and Microsporum gypseum. Hydroalcoholic extracts prepared with dried leaves from R. officinalis, Psidium guajava and T. riparia were assayed against dermatophyte species by the microdilution technique and by microscopy. R. officinalis and T. riparia were the most active against dermatophytes, as determined from the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC), and were investigated further. Fluorescence microscopy and scanning electron microscopy were used to investigate inhibition of hyphal growth by the two extracts, and showed a strong inhibition and an irregular growth pattern. Both extracts showed good action against dermatophytes, inhibiting fungal growth and causing alterations in their hyphae. Therefore, R. officinalis and T. riparia are potential sources of new compounds for the development of antifungal drugs.

Effects of fluconazole treatment of mice infected with fluconazole-susceptible and -resistant Candida tropicalis on fungal cell surface hydrophobicity, adhesion and biofilm formation.

BACKGROUND: The incidence of Candida tropicalis less susceptible to fluconazole (FLC) has been reported in many parts of the world. OBJECTIVES: The aim of this study was to examine the changes of putative virulence attributes of Candida tropicalis accompanying the development of resistance to FLC in vitro and in vivo. MATERIALS AND METHODS: A FLC-resistant strain (FLC-R) was obtained after sequential exposure of a clinical isolate FLC-sensitive (FLC-S) to increasing concentrations of the antifungal. The course of infection by both strains was analyzed in BALB/c mice. Analyses of gene expression were performed by real-time polymerase chain reaction PCR. The cell surface hydrophobicity, adhesion and biofilm formation were also determined. RESULTS: Development of resistance to FLC could be observed after 15 days of subculture in azole-containing medium. Overexpression of MDR1 and ERG11 genes were observed in FLC-R, and this strain exhibited enhanced virulence in mice, as assessed by the mortality rate. All mice challenged with the FLC-R died and FLC-treatment caused earlier death in mice infected with this strain. All animals challenged with FLC-S survived the experiment, regardless of FLC-treatment. Overall, FLC-R derivatives strains were significantly more hydrophobic than FLC-S strains and showed greater adherence and higher capacity to form biofilm on polystyrene surface. CONCLUSIONS: The expression of virulence factors was higher in FLC-R-C. tropicalis and it was enhanced after FLC-exposure. These data alert us to the importance of identifying microorganisms that show resistance to the antifungals to establish an appropriate management of candidiasis therapy.

Bacterial population of a two-phase anaerobic digestion process treating effluent of cassava starch factory.

Different groups of microorganisms in a two-phase anaerobic system were enumerated to evaluate the prevalence of specific groups and species. Total and fecal coliforms showed similar values both in acidogenic and methanogenic reactors. The fecal streptococci were 4-fold higher in the acidogenic reactor, when compared with those of the methanogenic reactor. As expected, no methane forming or sulfate reducing bacteria were found in the acidogenic reactor. The populations of methanogenic bacteria were dominated by a mixed population of straight to curved rods and multicellular filaments which strongly resembled members of the genus Methanosaeta. Seven prevalent species of facultative anaerobic gram-negative bacteria were identified as Previdencia alcalifacienciens, Providencia rettgeri, Enterobacter cloaceae, Citrobacter freundii, Klebsiella oxytoca, Proteus pennri and Yersinia enterocolitica. The species Pseudomonas aeruginosa, Stentophomonas maltophila and Acinotobacter iwoffi, were the most frequently isolated glucose non-fermenting gram-negative bacilli. Among these species, only P. aeruginosa was present in high number in each sample.

Microbial populations of an upflow anaerobic sludge blanket reactor treating wastewater from a gelatin industry.

The microbial populations of an upflow anaerobic sludge blanket reactor, used for treating wastewater from the gelatin industry, were studied by microbiological methods and phase-contrast and electron microscopy. Microscopy examination of the sludge showed a complex mixture of various rod-shaped and coccoid bacterial pluslong filaments and verymobile curved rods. In addition free-living anaerobic ciliates and flagellates were also observed. The trophic group population observed in decreasing order of dominance were hydrolytic and acetogenic at 10(6) and sulfate reducing and methanogenic at 10(5). The rate of methane production in anaerobic granular sludge cultivated in growth medium supplement with formate pressurized with H2:CO2 showed a significant increase in methane yield compared with theseed culture containingthe same substrate and atmosphere of N2:CO2. Similar rates of methane production were observed when the growth medium was supplemented with acetate pressurized either with H2:CO2 or N2:CO2. The number of total anaerobic bacteria at 10(7), fecal coliforms and total coliforms at 10(6), and fecal streptococci at 10(3) is based on colony counts on solid media. The four prevalent species of facultative anaerobic gram-negative bacteria that belong to the family of Enterobacteriaceae were identified as Escherichia coli, Esherichia fergusonii, Klebsiella oxytoca, and Citrobacter freundii. The species Aeromonas hydrophila, Aeromonas veronii, Acinetobacter iwoffi and Stenotrophomonas maltophila were the most frequently isolated glucose fermenting and nonfermenting gram-negative bacilli.

Further evidence of the trypanocidal action of eupomatenoid-5: confirmation of involvement of reactive oxygen species and mitochondria owing to a reduction in trypanothione reductase activity.

Our group assays natural products that are less toxic and more effective than available nitroheterocycles as promising therapeutic options for patients with Chagas disease. Our previous study reported the trypanocidal activity of eupomatenoid-5, a neolignan isolated from the leaves of Piper regnellii var. pallescens, against the three main parasitic forms of Trypanosoma cruzi. The present study further characterizes the biochemical and morphological alterations induced by this compound to elucidate the mechanisms of action involved in the cell death of T. cruzi. We show that eupomatenoid-5 induced oxidative imbalance in the three parasitic forms, especially trypomastigotes, reflected by a decrease in the activity of trypanothione reductase and increase in the formation of reactive oxygen species (ROS). A reduction of mitochondrial membrane potential was then triggered, further impairing the cell redox system through the production of more ROS and reactive nitrogen species. Altogether, these effects led to oxidative stress, reflected by lipid peroxidation and DNA fragmentation. These alterations are key events in the induction of parasite death through various pathways, including apoptosis, necrosis, and autophagy.

Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor.

In this paper, we describe the purification of an antiviral peptide from seeds of Sorghum bicolor L. by a procedure that included gel filtration, ion exchange, and high-performance liquid chromatography (HPLC) in a reversed-phase column. Its molecular weight, determined by chromatographic mobility on the Shim-pack DIOL-150 gel permeation column in HPLC, was found to be 2000Da. The peptide designated 2kD peptide strongly inhibited the replication of herpes simplex virus type 1 (HSV-1), dose-dependently, at 40-90% of the control level, after incubation with 10-50 microM of the peptide, with EC(50) and EC(90) values of 6.25 and 15.25 microM, respectively. The IC(50) value of the 2kD peptide against Vero cells was 250 microM. Pre-incubation of HSV-1 with various concentrations of the 2kD peptide showed dose-dependent cytopathic effects (CPE) reduction patterns at concentrations from 6.25 to 50 microM. The presence of the 2kD peptide before HSV-1 infections showed moderate inhibition of virus-induced CPE as compared to during or after infections, with EC(50) values of 12.5, 6.25, and 6.25 microM, respectively. Similar results were observed when the 2kD peptide was assayed against bovine herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, the 2kD peptide showed weak activity against poliovirus type 1, a non-enveloped virus. Taken together, these results indicate that the 2kD peptide was able not only to inhibit the initiation and the spread of infection, but also had an in vitro prophylactic effect against HSV-1 infection.

Purification and partial characterization of N-acetyl-beta-D-glucosaminidase from Tritrichomonas foetus.

Cells of Tritrichomonas foetus were suspended in buffer (0.1 M phosphate, 0.15 M NaCl, pH 7), sonicated for 2 min on ice, and centrifuged at low speed (500 g/40 min) at 4 degrees C. The resulting supernatant was centrifuged at 100,000 g for 30 min at 4 degrees C. The N-acetyl-beta-D-glucosaminidase activity as assayed by fluorimetric assay using 4-methylumbelliferil beta-D-N-acetylglucosamine (4MU-GlcNAc) was found predominantly (> 95%) in the supernatant. Isolation of the enzyme was achieved by a combination of gel filtration with ion-exchange chromatography. Non-denaturing gel electrophoresis indicated that N-acetyl-beta-D-glucosaminidase activity was present in two bands. When the two fluorescent bands were excised from the non-denaturing gel and rerun on denaturing 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis they exhibited two proteins with molecular masses of 40 and 45 kDa. The pH optimum is approximately 7.5 and the temperature optimum is approximately 37 degrees C.

Polysaccharide and glycolipid composition in Tritrichomonas foetus.

1. The polysaccharide and glycolipid composition in Tritrichomonas foetus was studied by paper, thin-layer and gas-liquid chromatographic analysis. 2. The carbohydrate components of the polysaccharide were glucose (47%), galactose (34%) and mannose (19%). N-acetylneuraminic acid was the sialic acid derivative characterized in the flagellate whole cells. 3. The sialic acid density was estimated as 2.7 x 10(7) residues/cell. 4. The long-chain base dihydrosphingosine, the carbohydrates galactose (67%), glucose (21%) and mannose (12%) as well as the fatty acids myristic (48%) and palmitic (52%) acids were characterized as components of the total glycolipids of T. foetus. 5. Total glycolipids were fractionated: a galactocerebroside and a ganglioside were identified.

Structural changes at the site of Tritrichomonas foetus-erythrocyte interaction.

Tritrichomonas foetus strongly agglutinates human erythrocytes, thus suggesting the occurrence of an adhesin associated with its surface. Adherence was observed immediately after mixing of the parasites with erythrocytes and the intensity increased for up to 30 min. Scanning electron microscopy examination of T. foetus-erythrocytes attachment showed that trichomonad cytoadherence took place mainly through their anterior and recurrent flagella. Ultrastructural observations showed that T. foetus contacts human red blood cells through punctual binding, inducing the separation between the two lipid monolayers of the parasite plasma membrane. This structural modification was also seen in freeze-fracture replicas where protrusions on the P and depressions on the E fracture faces were observed. No intramembranous particles, which mainly correspond to membrane integral proteins, were observed at the adhesion areas, indicating lateral mobility of integral membrane components involved in the appearance of the intramembranous particles. However, no changes was observed on the surface coat.

Purification and immunocytochemical localization of neuraminidase from Tritrichomonas foetus.

Lysis of Tritrichomonas foetus with a solution of the non-ionic detergent Triton X-114 at 0 degree C, followed by low-speed centrifugation, resulted in a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and an aqueous phase. Neuraminidase activity was mostly located in the detergent-insoluble pellet. When the parasites were incubated with bacterial phosphatidylinositol phospholipase C (PI-PLC) prior to detergent solubilization and phase separation neuraminidase activity was predominantly recovered in aqueous phase, rather than in the pellet and detergent phase. The molecular mass determined by gel permeation in high performance liquid chromatography (HPLC) and SDS-PAGE was 80,000 Da. Indirect immunofluorescence microscopy using polyclonal antibodies raised in rabbits against the purified neuraminidase, indicated that the enzyme is exposed on the cell surface. Previous treatment of the cells with PI-PLC significantly reduced antibody binding. Incubation of cryo-sections with the antibodies followed by detection using gold-labelled anti-rabbit IgG confirmed the presence of neuraminidase in the plasma membrane enclosing the cell body and flagella and in the membrane of vesicles preferentially located at the peripheral region of the protozoan.

Identification and localization of an adhesin on the surface of Tritrichomonas foetus.

Tritrichomonas foetus is a mucosal parasite of the urogenital-vaginal tract of cattle that strongly adheres to erythrocytes, which suggests that it presents an adhesin that recognizes red blood cells from different animal species and blood groups. In the present report we describe a cell-fractionation method for obtainment of a membrane fraction of T. foetus, which adhered to red blood cells. The T. foetus adhesin was obtained after parasite lysis and fractionation followed by ultracentrifugation, whereby a 100,000-g pellet fraction showed a strong hemagglutinating activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction, of erythrocyte ghosts, and of ghosts allowed to interact with the parasite membrane fraction revealed the presence of a 100-kDa protein as the putative adhesin. Polyclonal antibodies obtained in rabbits immunized with this protein recognized proteins of 100 and 90 kDa as determined by immunoblotting. Confocal laser scanning microscopy and transmission electron microscopy of cells incubated first in the presence of the antibody and subsequently in the presence of fluorescein- or gold-labeled goat anti-rabbit IgG showed labeling of the protozoan surface as well as of some cytoplasmic vesicles.